Real-time visualization of heterotrimeric G protein Gq activation in living cells
1 Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands
2 Nijmegen Centre for Molecular Life Sciences, Department of Biochemistry, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands
3 Department of Chemistry and Pharmaceutical Sciences, Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
4 Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany
BMC Biology 2011, 9:32 doi:10.1186/1741-7007-9-32Published: 27 May 2011
Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose.
In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus.
Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.